The BfpE protein of enteropathogenic E. coli (EPEC) is a representative of a family of cytoplasmic membrane proteins that participate in the biogenesis of type IV pili. It is hypothesized that BfpE is directly required for pilus formation and that it is a key component of a protein complex encoded by the bfp gene cluster. Experimental strategies are proposed to gain a better understanding of the structure and function of BfpE. A non-polar mutation in bfpE will be constructed, and the effect of this lesion on pilus production and bacterial adherence will be examined. A detailed topological analysis of BfpA will be carried out using phoA/lacZ reporter systems. The effects of mutations in the hydrophilic segments of BfpE will be determined to identify critical residues for pilus function. Three different methods: selection of suppressor of BfpE, cross linking, and use of the two-hybrid system, are proposed to study the interaction of BfpE with other proteins. The results of the proposed research should advance our knowledge about the mechanisms involved in the biogenesis of EPEC pili.